(Supported in part by NIH GM51532 to M. Koonce) Cytoplasmic dynein is a minus-end directed microtubule motor believed to be involved in organelle and vesicular intracellular transport and in some higher eukaryotes, in mitotic spindle function. This macromolecular protein complex is composed of a pair of heavy chains of 540 kDa, and 80-85 kDa intermediate chain and at least one light chain of approximately 58 kDa. Expression of a carboxy-terminal 380 kDa fragment of the heavy chain in Dictyostelium produces a protein that approximates the size and shape of a single mechanochemical head (Koonce, M.P. & Samso, M. Over expression of Cytoplasmic Dynein's Globular Head Causes a Collapse of the Interphase Microtubule Network in Dictyostelium. Molecular Biology of the Cell 7, 935-948, 1996). The fact that his truncated protein binds to microtubules in an ATP-sensitive manner allows us to decorate microtubules with the advantage that the absence of the stalk joining the 2 twin h eads will reduce steric restriction and hence gain direct information on the interaction of the dynein head and the microtubule wall. Preliminary results show that the decoration of microtubules does not occur in a helicoidal fashion, which would have allowed helical reconstruction. Thus, the best way to gain structural information on the microtubule-dynein head complex is electron tomography. In this project we will attempt a 3D reconstruction using automated electron tomography data collection on the IVEM. We have recorded preliminary tilt series on the Zeiss 910, but have mostly concentrated on the structure of the dynein alone, which is done using the single-particle approach. A paper on the 3-D structure of dynein has been submitted. Samso, M., Radermacher, M., Frank, F and Koonce, M.P. (1997) Structural Characterization of a Dynein Motor Domain. J. Mol. Biol. (submitted)